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1.
Six monoclonal antibodies (mabs) produced against Pasteurella piscicida whole cells, cultured under iron limitation conditions, were characterized and specificity tested. Two of the mabs (WE15H1D7 and WE3D6C11) appeared to be specific for P. piscicida lipopolysaccharide, while mabs WE14C10F9 and WE12D7D8 crossreacted with Photobacterium species. The remaining two mabs, WE9D6D8 and VP4B11, crossreacted with other bacterial genera. All mabs reacted with material present in the extracellular products of the pathogen as well as to whole cells, suggesting that the antigens to which antibodies are directed slough off the cell membranes or are secreted to the culture medium. The potential of the mabs as tools for the diagnosis of pasteurellosis is discussed.  相似文献   

2.
Abstract. Outer membranes and lipopolysaccharides of the marine fish pathogens Vibrio salmonicida and Vibrio anguillarum were isolated. SDS-PAGE profiles of purified LPS preparations from V. salmonicida revealed a broad low molecular weight band, whereas V. anguillarum LPS profiles demonstrated both a low-molecular band and several weaker high-molecular weight bands. Hydrolysis of V, salmonicida and V. anguillarum LPS separating the polysaccharide chain from the lipid A part and subsequent gel-chromatography suggests a polysaccharide molecular weight of ca. 1000 ('rough type' LPS) and ca. 6000 ('smooth type' LPS), respectively. Western blot of V. salmonicida outer membrane preparations and purified lipopolysaccharides and subsequent immunostaining with mouse monoclonal antibodies was performed. Eleven out of 15 monoclonal antibodies made against V. salmonicida cells reacted with one broad antigen-band in the low molecular weight region of both outer membrane and LPS profiles, corresponding to the LPS region. The previously reported outer surface antigen, VS-P1 from V. salmonicida , was observed to carry LPS epitopes as revealed by binding of monoclonal antibodies to VS-P1 as well as purified LPS preparations. These results strongly suggest that the VS-P1 antigen is a complex of both protein and LPS molecules.  相似文献   

3.
Abstract. Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Elcctrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguillarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LPS antigen band.  相似文献   

4.
Abstract. Atlantic salmon, Salmo salar L., responded to intraperitoneal injection of formalin killed Vibrio salmonicida or live infectious pancreatic necrosis virus ( ipnv ) by producing specific antibodies. The antibody titre varied significantly within the group tested. Western blot analysis demonstrated that high-titre antisera recognized two major bacterial antigens with molecular weights of 12–15 kD and 22–27 kD. In addition, a few narrow bands with higher molecular weights were observed. An antiserum raised against IPNV recognised two major antigens corresponding to the structural proteins of the virus. E lisa and Western blot analysis showed that the immune serum raised against Vibrio salmonicida reacted slightly with Vibrio anguillarum , whereas no reaction to Yersinia ruckeri or Aeromonas salmonicida was detected. Indirect elisa and an elisa competition assay revealed that the immune serum raised against the N1 serotype was specific for this serotype of ipnv . The results demonstrate that Atlantic salmon has a humoral immune system capable of producing antibodies which discriminate between related bacterial antigens and between different serotypes of a virus.  相似文献   

5.
Abstract. Extracellular hacmolytic activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the membrane-active enzyme glyccrophospholipidrcholcstcrol acyltransferase (GCAT). About 10% of the total haemolytic activity was due to a high molecular mass complex of LPS and GCAT (mol. mass >1000kDa), containing 35–50% neutral sugars and 1.5–2.0% protein. Some haemolytic activity (30–40% of total), corresponding to 50–70kDa by gel filtration, also contained GCAT-activity and may represent aggregated forms of GCAT. However, about 50% or more of the haemolytie activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the hacmolytic activity of both GCAT and GCAT-LPS. A transposon-produccd serinc protease negative mutant of the same A. salmonicida strain showed reduced haemolytic activity. The mutant produced a 38-kDa GCAT proform of low hacmolytic activity. The proform was processed by autogenous scrinc protease to a highly hacmolytic 26-kDa molecule with pl 6.3, similar to GCAT of the parent strain. The weakly haemolytic GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.  相似文献   

6.
Atlantic salmon were vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa) by injection with three vaccines developed in our laboratory and an autogenous bacterin (IcelandBiojec.OO, IBOO) produced by a commercial vaccine producer. The humoral antibody responses to bacterial antigens were monitored by ELISA and Western blotting. The fish were challenged by infection with Asa 6 and 12 weeks post-vaccination. Protection was induced in all groups of vaccinated fish. The protection achieved was time-dependent. The autogenous bacterin, IBOO, induced a protective immune response later than our experimental vaccines. All the vaccines tested induced specific antibody response that increased between 6 and 12 weeks after vaccination. The antibody response was mainly directed against the A-layer protein, but antibodies to other bacterial components were also detected. Significant correlation was obtained between the antibody titre to extracellular Asa antigens, induced by the different vaccine preparations, and survival of vaccinated fish challenged by a virulent Asa strain. Furthermore, the detection of antibodies directed against an extracellular toxic metallo-caseinase, AsaP1, in fish sera correlated with protection.  相似文献   

7.
Abstract. Controversy exists concerning the efficacy of vaccinating fish against furunculosis. Where success is claimed, there has been little attempt to characterize the protective antigens or confirm their immunogenicity. In this report, the immunogenicity of native extracellular products (ECP) of Aeromonas salmonicida and a formalin-inactivated toxoid of ECP (f-ECP) was studied in rainbow trout and rabbits, with particular attention to the putative bacterial virulence factors protease and haemolysin. Using crossed immunoelectrophoresis and Protein-A absorption, antibodies to seven ECP components were detected in the rabbit following immunization with native ECP; antihaemolysin antibodies were found but antibodies to the protease could not be detected. Antibodies to at least 14 components of ECP, including haemotysin and protease, were detected in the rabbit following immunization with f-ECP. In trout immunized either with native ECP or f-ECP, antibodies to only four ECP antigens were detected and no antibodies to haemolysin or protease were found. The results may explain previous reports that passive immunization with rabbit antisera gave superior protection against furunculosis compared with antisera raised in fish, and indicate that many extracellular antigens of A. salmonicida may require modification in order to improve their immunogenicity in fish.  相似文献   

8.
Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.  相似文献   

9.
Abstract. Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.  相似文献   

10.
Abstract. A cell surface product (VS-P1) of Vibrio salmonicida has been purified from culture supernatant by a combination of extensive dialysis, filtration and centrifugation, as well as by salt precipitation and hydrophobic chromatography. SDS-PAGE analysis showed that the monomeric form of the antigen is a single polypeptide with an apparent molecular weight of 40000. Size exclusion HPLC of purified VS-P1 as well as VS-Pl-containing fish serum revealed, however, oligomeric forms in the range from 300000 to more than 700000 daltons. The antigen contained 6% carbohydrate and several isolectric forms were distinguishable when analysed on an analytical isoelectric focusing electrophoresis system. A 'sandwich' ELISA, utilizing polyclonal antibodies, was developed for screening sera from both healthy and moribund Atlantic salmon for the presence of the VS-P1 antigen.  相似文献   

11.
Abstract. Monoclonal antibodies (Mab) directed against Vibrio salmonicida were produced and partially characterized. The bacterium is the causative agent of 'Hitra disease' or cotdwater vibriosis (CV) and differs from all other Vibrio bacteria tested so far with respect to a unique surface antigen (VS-P1). Thirteen hybridoma clones produced antibodies which exclusively reacted with this antigen in ELISA. The remaining four clones reacted against undefined determinants and were partly cross-reactive to V. anguiilarum, V. ordalii and V. fischeri . Fifteen Mab were of IgG1/kappa and two of the IgG3/kappa isotypes. Eleven of the IgG1 plus the two IgG3 Mab reacted with the VS-P1 molecule.  相似文献   

12.
Abstract. Two haemolysin genes (AHH4 and AHH-2) of Aeromonas hydrophila ATCC7966 were cloned into a plasmid vector in Escherichia coli K-12. An open reading frame (ORF) of the AHH-1 haemolysin gene was 1734 base pairs (bp). and corresponded to a protein of 577 amino acid residues. Analysis of the deduced amino sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The sequence also included the -10 region and the -35 region of a promoter, and a ribosome- binding site upstream from the ORF. The termination site was located downstream from the ORF. The haemolysin was a thermolabile protein with the predicted molecular mass of 60 kDa. The AHH-1 gene is distributed in various A. hydrophila and A. salmonicida strains. The nucleotide sequence of a 981 bp ORF of the AHH-2 gene was encoded with the predicted molecular mass of 377 kDa polypeptides. The homology of the nucleotide sequence was very low between the AHH-1 and AHH-2 genes, and also with the aerolysin gene cloned by Howard & Buckley (19S6). No leader peptide was found in the N-terminal region of the ORF of the AHH2 gene. The AHH-2 gene was detected in the original strain ATCC7966, but was not detected in other tested strains of A. hydrophila and A. salmonicida.  相似文献   

13.
Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn2+ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.  相似文献   

14.
Abstract. Extracellular product (ECP) antigens of Aeromonas salmonicida were modified in an attempt were tested on an antigen-induced proliferation assay, for the ability to induce antibodies as measured by dot blot dot assay and as vaccines in vaccination/challenge trials. Modifications tested included particularization on to polystyrene beads, coating on to sheep red blood cells, mixing with BCG vaccine as adjuvant, and attachment to the T-independent carrier Fieoll. The only modification that resulted in increased protection levels was the particularization on to polystyrene beads.  相似文献   

15.
In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.  相似文献   

16.
Abstract. Mutants of Aeromonas salmonicida strains lacking either the A-protein, O-antigen or both of these major surface antigens were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), for their suitability as live vaccines (LV). All of these mutants were shown to be attenuated, as fish receiving ∼5 × 107 of the respective strains showed no clinical signs of furunculosis. Immersion vaccination of fish in 5 × 107 cfu ml-1 of these strains with an identical immersion dose 14 days later resulted in significant protection by all strains from challenge with a heterologous virulent strain of A. salmonicida 5 weeks later. The levels of protection conferred were all greater than or equal to that provided by an injected bacterin using the same vaccination schedule. With one exception, all LV strains that still possessed a functional O-antigen provided protective indices (PI) four- to seven-fold greater than the PI for the fish injected with bacterin. When antibody responses of vaccinated fish were compared, it was found that only vaccination by bacterin gave rise to a measurable agglutinating litre. Western immunoblots using the immune fish sera failed to reveal any major differences in antigen recognition in fish that received any of the vaccines tested. These data suggest that the immune response generated by the use of live vaccine strains is different from that generated by a bacterin, and that these useful mutations may be incorporated into existing furunculosis LVs for further attenuation.  相似文献   

17.
Abstract. The putative virulence factors of Aeromonas salmonicida , the aetiological agent of furunculosis in salmonids, are candidates for protective antigens in effective vaeeines against furunculosis. In this report, the authors have compared the immunogenieily of eell-associated and extracellular antigens of A. salmonicida in Atlantic salmon, Salmo salar L., to that in rabbit. The animals were immunized with formalin-killed whole cells and formalin-inactivated extracellular products (ECP), either separately or in combination. The ability of the antigens to induce antibody production was studied by elisa and Western blotting techniques. These results confirm previous reports that far more structures are immunogenie in rabbit compared to the antibody responses elicited in salmon. However, in both species, some antigens were dominant, including a caseinolytic protease in addition to the A-protein and high and low MW LPS.  相似文献   

18.
Abstract. The predominant cell surface protein (A-protein) of Aeromonas salmonicida has been purified by a method utilizing a glycine/hydrochloridc extraction from whole cells and HPLC/ion exchanger (DEAE) columns. This procedure yielded two LPS-frec molecules (a 40- and a 50-kDa form) both shown to contain A-protein determinants. The former appears to be a digest product of the latter, as a serine protease produced by A. salmonicida was shown to process the 50-kDa form into a 40-kDa molecule in vitro. The A-layer protein was shown to contain one isoform, although multiple isoelectric forms appeared as preparative artifacts, probably due to deamidation. The A-layer protein and LPS arc the most significant surface antigens recognized by the Atlantic salmon B-lymphocytes or antibodies. Immunological studies of LPS-free and LPS-containing A-protein preparations were undertaken to test whether the two components behave like antigenie competitors or whether the LPS moiety could adjuvant the antibody response against the A-protein. The latter was shown to be the case.  相似文献   

19.
Abstract. A collection of 130 strains of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida isolated from diseased salmonids in Denmark, Norway, North America and Scotland has been characterized with regard to protein patterns, antibiotic resistance and exoprotease activity. Whole cell and outer membrane protein profiling could distinguish three different profiles in A. salmonicida. Eight outer membrane proteins were demonstrated (49, 40, 38, 37, 33, 31, 30 and 29 kDa). One protein profile was deficient in a 38 kDa outer membrane protein and instead contained an outer membrane protein of 37 kDa which was not detectable among the other protein profiles. Strains with the 37 kDa outer membrane protein showed multiple low-level antibiotic resistance towards cephalothin, penicillin, chloramp-henicol, tetracycline and quinolones. In addition, these strains were exoprotease deficient. Strains with the 37 kDa protein were unable to degrade cattle and trout serum proteins and displayed a delayed degradation of casein. Haemolysis on cattle blood agar plates was similarly delayed. In vivo examination of extracellular products from a normal protein profile strain and one with the 37 kDa outer membrane protein demonstrated major differences in pathological effects in rainbow trout. The strain possessing the 37 kDa outer membrane protein produced almost no pathological effects while the normal protein profile strain produced typical furuncles.  相似文献   

20.
Four non-pigment-producing isolates and two pigment-producing isolates of Aeromonas salmonicida sp. salmonicida were isolated from the head-kidney of diseased farmed Atlantic salmon, Salmo salar L. The cultural, morphological and biochemical features of the isolates were compared with those of reference strains. Injection and cohabitation experiments were performed. The only difference between the non-pigment-producing isolates and the pigment producing reference strains of A. salmonicida ssp. salmonicida was the inability of the former to produce pigment. In the injection experiments, the investigated non-pigment-producing isolate produced a significantly higher mortality compared with the mortality caused by the reference strain, whereas no difference in mortality was detected in the cohabitation experiments.  相似文献   

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